Genomic divergence and phenotypic heterogeneity of Legionella longbeachae reveal a putative novel proinflammatory serogroup with genome reduction and mobilome expansion

ABSTRACT Legionella longbeachae is an emerging respiratory pathogen primarily found in soil environments, yet its genomic architecture and evolutionary strategies remain poorly characterized. Here, through pan-genomic and functional analysis of 242 L. longbeachae isolates, we reveal an open pan-genome driven by extensive horizontal gene transfer and marked functional divergence between core and accessory genomes. We identify a phylogenetically distinct lineage, designated as putative serogroup 3 (sg3), recovered from Chinese environments. This lineage diverges from canonical serogroups 1 and 2 through genome reduction (mean size 4.01 Mb), absence of plasmids, and a truncated O-antigen biosynthesis cluster lacking a key N-acetyltransferase-encoding gene (orf9). In vitro infection models across multiple human cell lines show that sg3 elicits increased host cell death and elevated proinflammatory cytokine transcription in epithelial cells compared to sg1 and sg2. Phylogenetically corrected association analyses revealed no significant link between this phenotype and individual accessory virulence factors. Instead, rank-transformed phylogenetically generalized least squares (PGLS) regression demonstrated a significant positive association with expansion of the mobilome (COG X), while intracellular trafficking (COG U) and defense (COG V) repertoires were contracted. These findings reveal a distinct pathogenic profile in sg3 that differs from the canonical serogroups, characterized by heightened inflammatory activation rather than immune evasion. Our work advances the understanding of L. longbeachae population structure, challenges the reductionist utility of traditional serogrouping, and highlights the potential need for revised diagnostic considerations as well as continued surveillance of lineages displaying enhanced cytotoxicity and proinflammatory responses, which may be associated with mobilome expansion. IMPORTANCE Legionella longbeachae is an understudied yet emerging cause of Legionnaires’ disease, with a distinct soil-based ecology. Using comparative pan-genomics and functional infection assays across 242 isolates (including 39 newly sequenced from China), we identify a putative novel serogroup (sg3) that has undergone marked genome reduction (4.01 Mb) and completely lost plasmids, contrasting with near-ubiquitous plasmid carriage in sg1/sg2. Despite its streamlined genome, putative sg3 exhibits enhanced cytotoxicity in all tested human cell lines and elevated proinflammatory cytokine expression, particularly in epithelial cells. These phenotypes are not explained by individual virulence genes but are significantly associated with mobilome (COG X) expansion. Our findings question the assumption that genome reduction necessarily diminishes pathogenic potential and suggest that higher-order genomic restructuring, potentially linked to mobilome expansion, may be associated with increased inflammatory responses. Furthermore, they highlight the potential need for continued surveillance and consideration of revised diagnostic approaches to include this rare but possibly underdiagnosed lineage.